DNA Fingerprinting of Pearls to Determine Their Origins

We report the first successful extraction of oyster DNA from a pearl and use it to identify the source oyster species for the three major pearl-producing oyster species Pinctada margaritifera, P. maxima and P. radiata. Both mitochondrial and nuclear gene fragments could be PCR-amplified and sequenced. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay in the internal transcribed spacer (ITS) region was developed and used to identify 18 pearls of unknown origin. A micro-drilling technique was developed to obtain small amounts of DNA while maintaining the commercial value of the pearls. This DNA fingerprinting method could be used to document the source of historic pearls and will provide more transparency for traders and consumers within the pearl industry

Regionalized Pathology Correlates with Augmentation of mtDNA Copy Numbers in a Patient with Myoclonic Epilepsy with Ragged-Red Fibers (MERRF-Syndrome)

Human patients with myoclonic epilepsy with ragged-red fibers (MERRF) suffer from regionalized pathology caused by a mutation in the mitochondrial DNA (m.8344A→G). In MERRF-syndrome brain and skeletal muscles are predominantly affected, despite mtDNA being present in any tissue. In the past such tissue-specificity could not be explained by varying mtDNA mutation loads. In search for a region-specific pathology in human individuals we determined the mtDNA/nDNA ratios along with the mutation loads in 43 different post mortem tissue samples of a 16-year-old female MERRF patient and in four previously healthy victims of motor vehicle accidents. In brain and muscle we further determined the quantity of mitochondrial proteins (COX subunits II and IV), transcription factors (NRF1 and TFAM), and VDAC1 (Porin) as a marker for the mitochondrial mass. In the patient the mutation loads varied merely between 89–100%. However, mtDNA copy numbers were increased 3–7 fold in predominantly affected brain areas (e.g. hippocampus, cortex and putamen) and in skeletal muscle. Similar increases were absent in unaffected tissues (e.g. heart, lung, kidney, liver, and gastrointestinal organs). Such mtDNA copy number increase was not paralleled by an augmentation of mitochondrial mass in some investigated tissues, predominantly in the most affected tissue regions of the brain. We thus conclude that “futile” stimulation of mtDNA replication per se or a secondary failure to increase the mitochondrial mass may contribute to the regionalized pathology seen in MERRF-syndrome

Human native lipoprotein-induced de novo DNA methylation is associated with repression of inflammatory genes in THP-1 macrophages

<p>Abstract</p> <p>Background</p> <p>We previously showed that a VLDL- and LDL-rich mix of human native lipoproteins induces a set of repressive epigenetic marks, <it>i.e. de novo </it>DNA methylation, histone 4 hypoacetylation and histone 4 lysine 20 (H4K20) hypermethylation in THP-1 macrophages. Here, we: 1) ask what gene expression changes accompany these epigenetic responses; 2) test the involvement of candidate factors mediating the latter. We exploited genome expression arrays to identify target genes for lipoprotein-induced silencing, in addition to RNAi and expression studies to test the involvement of candidate mediating factors. The study was conducted in human THP-1 macrophages.</p> <p>Results</p> <p>Native lipoprotein-induced <it>de novo </it>DNA methylation was associated with a general repression of various critical genes for macrophage function, including pro-inflammatory genes. Lipoproteins showed differential effects on epigenetic marks, as <it>de novo </it>DNA methylation was induced by VLDL and to a lesser extent by LDL, but not by HDL, and VLDL induced H4K20 hypermethylation, while HDL caused H4 deacetylation. The analysis of candidate factors mediating VLDL-induced DNA hypermethylation revealed that this response was: 1) surprisingly, mediated exclusively by the canonical maintenance DNA methyltransferase DNMT1, and 2) independent of the Dicer/micro-RNA pathway.</p> <p>Conclusions</p> <p>Our work provides novel insights into epigenetic gene regulation by native lipoproteins. Furthermore, we provide an example of DNMT1 acting as a <it>de novo </it>DNA methyltransferase independently of canonical <it>de novo </it>enzymes, and show proof of principle that <it>de novo </it>DNA methylation can occur independently of a functional Dicer/micro-RNA pathway in mammals.</p

Botulinum Neurotoxin D Uses Synaptic Vesicle Protein SV2 and Gangliosides as Receptors

Botulinum neurotoxins (BoNTs) include seven bacterial toxins (BoNT/A-G) that target presynaptic terminals and act as proteases cleaving proteins required for synaptic vesicle exocytosis. Here we identified synaptic vesicle protein SV2 as the protein receptor for BoNT/D. BoNT/D enters cultured hippocampal neurons via synaptic vesicle recycling and can bind SV2 in brain detergent extracts. BoNT/D failed to bind and enter neurons lacking SV2, which can be rescued by expressing one of the three SV2 isoforms (SV2A/B/C). Localization of SV2 on plasma membranes mediated BoNT/D binding in both neurons and HEK293 cells. Furthermore, chimeric receptors containing the binding sites for BoNT/A and E, two other BoNTs that use SV2 as receptors, failed to mediate the entry of BoNT/D suggesting that BoNT/D binds SV2 via a mechanism distinct from BoNT/A and E. Finally, we demonstrated that gangliosides are essential for the binding and entry of BoNT/D into neurons and for its toxicity in vivo, supporting a double-receptor model for this toxin

The Polarity Protein Scribble Regulates Myelination and Remyelination in the Central Nervous System

The development and regeneration of myelin by oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), requires profound changes in cell shape that lead to myelin sheath initiation and formation. Here, we demonstrate a requirement for the basal polarity complex protein Scribble in CNS myelination and remyelination. Scribble is expressed throughout oligodendroglial development and is up-regulated in mature oligodendrocytes where it is localised to both developing and mature CNS myelin sheaths. Knockdown of Scribble expression in cultured oligodendroglia results in disrupted morphology and myelination initiation. When Scribble expression is conditionally eliminated in the myelinating glia of transgenic mice, myelin initiation in CNS is disrupted, both during development and following focal demyelination, and longitudinal extension of the myelin sheath is disrupted. At later stages of myelination, Scribble acts to negatively regulate myelin thickness whilst suppressing the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAP) kinase pathway, and localises to non-compact myelin flanking the node of Ranvier where it is required for paranodal axo-glial adhesion. These findings demonstrate an essential role for the evolutionarily-conserved regulators of intracellular polarity in myelination and remyelination

GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white matter injury.

Diffuse white matter injury (DWMI), a leading cause of neurodevelopmental disabilities in preterm infants, is characterized by reduced oligodendrocyte formation. NG2-expressing oligodendrocyte precursor cells (NG2 cells) are exposed to various extrinsic regulatory signals, including the neurotransmitter GABA. We investigated GABAergic signaling to cerebellar white matter NG2 cells in a mouse model of DWMI (chronic neonatal hypoxia). We found that hypoxia caused a loss of GABAA receptor-mediated synaptic input to NG2 cells, extensive proliferation of these cells and delayed oligodendrocyte maturation, leading to dysmyelination. Treatment of control mice with a GABAA receptor antagonist or deletion of the chloride-accumulating transporter NKCC1 mimicked the effects of hypoxia. Conversely, blockade of GABA catabolism or GABA uptake reduced NG2 cell numbers and increased the formation of mature oligodendrocytes both in control and hypoxic mice. Our results indicate that GABAergic signaling regulates NG2 cell differentiation and proliferation in vivo, and suggest that its perturbation is a key factor in DWMI

Addressing food and nutrition insecurity in the Caribbean through domestic smallholder farming system innovation

Structural conditions underlying the development of CARICOM’s two-tiered agricultural innovation system depict diverse drivers of change over time, versus institutional inertia of export-oriented formal institutions and the neglect of informal domestic markets. Key principles of taking an agroecological approach would include: supporting diversity and redundancy, building connectivity, managing slow variables and feedbacks, improving understanding of socioecological systems as complex adaptive systems, and encouraging polycentric governance systems. In this paper, we review the conditions that have been undermining sustainable food and nutrition security in the Caribbean, focusing on issues of history, economy, and innovation

Nonrandom Selection and Multiple Blood Feeding of Human Hosts by<i> Anopheles</i> Vectors: Implications for Malaria Transmission in Papua New Guinea

Nonrandom selection and multiple blood feeding of human hosts by Anopheles mosquitoes may exacerbate malaria transmission. Both patterns of blood feeding and their relationship to malaria epidemiology were investigated in Anopheles vectors in Papua New Guinea (PNG). Blood samples from humans and mosquito blood meals were collected in villages and human genetic profiles ("fingerprints") were analyzed by genotyping 23 microsatellites and a sex-specific marker. Frequency of blood meals acquired from different humans, identified by unique genetic profiles, was fitted to Poisson and negative binomial distributions to test for nonrandom patterns of host selection. Blood meals with more than one genetic profiles were classified as mosquitoes that fed on multiple humans. The age of a person bitten by a mosquito was determined by matching the blood-meal genetic profile to the villagers' genetic profiles. Malaria infection in humans was determined by PCR test of blood samples. The results show nonrandom distribution of blood feeding among humans, with biased selection toward males and individuals aged 15-30 years. Prevalence of Plasmodium falciparum infection was higher in this age group, suggesting males in this age range could be super-spreaders of malaria parasites. The proportion of mosquitoes that fed on multiple humans ranged from 6% to 13% among villages. The patterns of host utilization observed here can amplify transmission and contribute to the persistence of malaria in PNG despite efforts to suppress it with insecticidal bed nets. Excessive feeding on males aged 15-30 years underscores the importance of targeted interventions focusing on this demographic group